PONE-D-21-31062Comparison of six methods for Loa loa genomic DNA extractionPLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract (perhaps include in the main manuscript next time for easier reviewing)

Why is DNA extraction linked with vaccine delivery here? (read further, change to development)

When you get down to the results section, it’s a lot of numbers and repeats of A260/A280. Perhaps think of a way to present without just having a wall of numbers.

Introduction

No need to put (L. loa) in brackets, just leave it at Loa loa and next mention L. loa.

Line 50: rather than name checking Qiagen here, I would just state that commercial DNA extraction kits exist. Obviously there are many of these from different companies, I wouldn’t put a specific kit here in the introduction. Qiagen is likely the most expensive kit – a consideration when it comes to thinking about wide spread use in low and middle income areas where parasites are often highly endemic.

Methods

Presumably the microfilariae came from human participants? Need to put in a little about how infected individuals were selected, consent forms and ethics, and blood collection.

Line 77: So there were 12 tubes in total each with 350,000 microfilariae in them? 2 for each technique?

Line 84: Put a reference in for Barker’s procedure

Line 86: Lysed how? Mechanical? Vortex? Heat?

Line 92: Stored dry or resuspended in buffer or water?

Line 95: Perhaps include a ref or link to the kit protocol – and state which protocol as most kits have a few different protocols in the manual depending on what material you have

Line 99: space between 5C°for

Line 100: space between number and units

Line 107: as per above procedure, stored dry or in

Line 112: reference for Miller

Tris-EDTA and methanol – these are quite crude extracts, any issues with inhibition in down stream applications (i.e. pcr)?

Line 166: Amplification of the extracted DNA? Either way, please ad in the PCR details (ah I see this is down further, perhaps switch the electrophoresis and PCR sections around). A real-time PCR would be interesting to check on efficiency and potential inhibition etc

Line 179/180: space between number and unit

Line 184: Where did the primers come from? Reference to paper they were first reported in. What is the target gene?

Discussion

Line 284: How was the contamination identified? What makes you certain it is salt and not something else foreign introduced into the prep?

Line 309: Indicating what activity?

It would be much easier to read the discussion if you could refer to tables or figures where the results are. Just stating yield was higher without the actual yield or reference to where the yield information is, isn’t very informative.

Line 311: Band intensity is not a great measure for DNA concentration. Particularly when doing it by eye.

Line 314: Distortion on the pcr amplicon, after endonuclease, just genomic DNA? Need to be clearer when talking about gel results as to which sample you are talking about.

Line 323: here you state the DNA concentration (by only one method, qubit?) for Tris-EDTA, and lower down for methanol. But it is not stated above for salting out, Qiagen or phenol/chloroform methods. Just a relative, higher or lower DNA. Refer to the table.

Line 338: Presumably for the PCR amplicon as just extracted genomic DNA would appear as a smear.

Line 352: Despite the band distortion? Based on Figure 3 I would not be recommending salting out unless some optimization/post DNA extraction step is taken to clean it up. I also wouldn’t recommended it on a time basis (table 1) as it didn’t work optimally, and it takes a long time to do. It’s only good point is that it is cheap. Normally you would say 2/3 – the three being time, cost, purity. Salting out is 1/3. At nest.

Was there any reason to use Qiagen as the commercial kit and not another, perhaps less expensive, commercial kit?

Figures: I know this is probably journal requirements, but I hate not having figure captions with the figures.

You should also be referring to these more often in the discussion – wherever you mention gel results, refer to the figure that shows it. Ditto the table.

2A and 2B are the replicates? Perhaps add that in the captions, Fig 2A replicate one for each extraction procedure pre and post endonuclease digestion etc.

Table: This appears to only be one of the replicates for each DNA extraction? Would like to see data for both replicates. Also is this Nanoview plus or Qubit results? Or both combined?

Reviewer #2: This is a relatively simple and straightforward report on comparing DNA extraction techniques for the diagnosis of an important human filarial nematode, Loa loa. Sensitive DNA extraction is key for diagnostics for this disease since many patients have low microfilaria concentrations, making diagnosis and appropriate treatment difficult. The authors compare six different extraction methods varying in cost and efficacy. Because cost and safety of use in the field is critical, this is an important contribution. The finding that the simple 'salting-out' method provides sufficient quality DNA for diagnostic tests and further evaluations is important and significant. Although not the most rapid method, it was highly efficient and could lead to more accurate diagnostics in the field.

I found this manuscript easy to follow for the most part. However, the Results section on Spectrometry Analysis was confusing and needs to be rewritten. In this section, the authors report a 260/280 ration for salting out as 1.9, yet in Table 1 they report 2.01, which is high and indicates contamination. The further analyses suggest the DNA extracted by 'salting-out' is of satisfactory quality for manipulation and diagnostics. There are other inconsistencies; they indicate on L197 that concentrations were 'far behind those of' when I think they mean 'far greater than.' Much of this text is redundant to Table 1, so perhaps the authors can combine the first two sections into one, entitled 'Comparative spectrometric analysis of the six methods'.

Overall, a very useful and practical contribution with the clarifications indicated above.

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Comparison of six methods for Loa loa genomic DNA extraction

PONE-D-21-31062R1

Dear Dr. Akue,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No